Simian virus 40 (SV40) has two early transcriptional units that are transcribed from a region near the origin of replication. The early-early (EE) transcription unit, whose RNA encodes a large T- antigen and small t-antigen, predominates at early times post infection. The late-early (LE) transcription unit RNA initiation sites are located upstream of the EE TATA box and function at late time post infection. We have characterized the in vitro translational efficiency of SV40 early-early (EE) and two late- early (LE) RNAs. We demonstrated that the presence of one or two potential AUG initiator codons in the leader sequences of the LE RNAs inhibits efficient translation from the downstream T- antigen initiator, AUG. In addition, translation of the LE RNA resulted in the synthesis of new viral proteins, 2.7 Kd in size. The role of this protein is currently under investigation. Carboxy terminal mutants of T-antigen cause a minimal decrease in the efficiency of viral DNA replication but under appropriate conditions significantly decrease the yield of infectious virus particles by three orders of magnitude. Our studies have demonstrated that a reduction in viral late RNA is, in part, responsible for the lower titers produced by these mutants in CV- 1P cells. Furthermore, we have demonstrated that the viral late protein, agnoprotein, is not produced in CV-1P cells infected with C-terminal mutants. This suggest that T-antigen plays a role in the translation of agnoprotein.